Enabling Protein Degradation Drug Discovery

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  • Name
    Catalogue Number
    Size
    Price
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  • Name:
    TRIAD1 [6His-tagged]
    Catalogue Number:
    63-0029-025
    Size:
    25 µg
    Price:
    £175
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  • Species
    Human
  • Source
    Sf21 insect cell-baculovirus expression
  • Quantity
    25 μg
  • Storage
    -70°C
  • Concentration
    0.5 mg/ml
  • Formulation
    50 mM HEPES pH 7.5, 150 mM sodium chloride, 2 mM dithiothreitol, 10% glycerol
  • Molecular Weight
    ~61.2 kDa
  • Stability
    12 months at -70°C; aliquot as required
  • Protein Sequence
    Accession number: NP_006312. For full protein sequence information download the Certificate of Analysis pdf.
  • QA; Protein Identification
    Confirmed by mass spectrometry.
  • QA; Activity
    E3 ligase assay: The ubiquitin conjugating activity of His-TRIAD1 was validated through its ability to catalyse the generation of polyubiquitin chains in the presence of the E1 activating enzyme His-UBE1, the E2 conjugating enzyme His-UBE2D1 (UbcH5a) (several E2s were tested, data generated with this E2 is provided by way of example) and ubiquitin. Incubation of His-TRIAD1 for 120 minutes at 37°C in the presence of ubiquitin, His-UBE1, His-UBE2D1 and ATP (Lane 1) was compared alongside two control reactions with either ATP (Lane 2) or His-TRIAD1 (Lane 3) excluded from the reaction. Ubiquitin conjugates were identified by Western blotting using an anti-ubiquitin conjugate antibody and these were observed only in the presence of both ATP and His-TRIAD1.

The enzymes of the ubiquitylation pathway play a pivotal role in a number of cellular processes including the regulated and targeted proteasome-dependent degradation of substrate proteins. Three classes of enzymes are involved in the process of ubiquitylation; activating enzymes (E1s), conjugating enzymes (E2s) and protein ligases (E3s). Triad Domain-Containing Protein 1 (TRIAD1) is a member of the E3 protein ligase family and cloning of the human gene was first described by van der Reijden et al. (1999). TRIAD1 contains a TRIAD motif containing two RING domains which flank a conserved cysteine-rich (C6HC) domain designated DRIL (double RING finger-linked domain) (Marteijn et al., 2005). TRIAD is thought to be involved in protein translation, interacting with UbcH7 (UBE2L3) to polyubiquitylate eIF4E2 targeting it for proteasomal degradation (Tan et al., 2003). More recently these proteins have been referred to as Ring in-between Ring E3 ligases (RBRs) that function like RING-HECT hybrids regulating processes such as translation and immune signalling (Wenzel et al., 2011). 

References:

Marteijn JA, van Emst L, Erpelinck-Verschueren CA, Nikoloski G, Menke A, de Witte T, Lowenberg B, Jansen JH, van der Reijden BA (2005) The E3 ubiquitin-protein ligase Triad1 inhibits clonogenic growth of primary myeloid progenitor cells. Blood 106, 4114-23.

Tan NG, Ardley HC, Scott GB, Rose SA, Markham AF, Robinson PA (2003) Human homologue of ariadne promotes the ubiquitylation of translation initiation factor 4E homologous protein, 4EHP. FEBS Lett 554, 501-4.

van der Reijden BA, Erpelinck-Verschueren CA, Lowenberg B, Jansen JH (1999) TRIADs: a new class of proteins with a novel cysteine-rich signature. Protein Sci 8, 1557-61.

Wenzel DM, Lissounov A, Brzovic PS, Klevit RE (2011) UBCH7 reactivity profile reveals parkin and HHARI to be RING/HECT hybrids. Nature 474, 105-8.